Chemical compositions of Eucalyptus sp. Essential oils and the evaluation of their combinations as a promising treatment against ear bacterial infections

Background The chemical composition and biological activities of Eucalyptus essential oils (EOs) have been documented in numerous studies against multiple infectious diseases. The antibacterial activity of individual Eucalyptus EOs against strains that cause ear infections was investigated in our previous study. The study's antibacterial activity was promising, which prompted us to explore this activity further with EO blends. Methods We tested 15 combinations (9 binary combinations and 6 combinations of binary combinations) of Eucalyptus EOs extracted by hydrodistillation from eight Tunisian Eucalyptus species dried leaves against six bacterial strains responsible for ear infections: three bacterial isolates (Haemophilus influenzae, Haemophilus parainfluenzae, and Klebsiella pneumoniae) and three reference bacteria strains (Pseudomonas aeruginosa, ATTC 9027; Staphylococcus aureus, ATCC 6538; and Escherichia coli, ATCC 8739). The EOs were analyzed using GC/FID and GC/MS. The major compounds, as well as all values obtained from the bacterial growth inhibition assay, were utilized for statistical analysis. Results The antibacterial activity of the EO blends exhibited significant variation within Eucalyptus species, bacterial strains, and the applied methods. Principal component analysis (PCA) and hierarchical cluster analysis (HCA), based on the diameters of the inhibition zone, facilitated the identification of two major groups and ten subgroups based on the level of antibacterial activity. The highest antibacterial activity was observed for the mixture of EOs extracted from E. panctata, E. accedens, and E. cladoclayx (paac) as well as E. panctata, E. wandoo, E. accedens, and E. cladoclayx (pwac) using the disc diffusion method. Additionally, significant activity was noted with EOs extracted from E. panctata, E. wandoo (pw) and E. panctata, E. accedens (pa) using the broth microdilution method. Conclusion Our findings suggest that certain EO combinations (paac, pwac, pw, and pa) could be considered as potential alternative treatment for ear infections due to their demonstrated highly promising antibacterial activities. Supplementary Information The online version contains supplementary material available at 10.1186/s12906-024-04494-2.


Background
Ear infections stand out as the most prevalent ailment across all age groups, predominantly impacting both adults and young children [1].Common bacterial strains associated with ear infections include Streptococcus pneumoniae, Staphylococcus aureus, Hemophilus influenzae, and Moraxella catarrhalis.Acute otitis media is typically linked to these strains, while chronic otitis media is more frequently associated with Pseudomonas aeruginosa, S. aureus, Proteus mirabilis, Klebsiella pneumoniae, and Escherichia coli.These bacteria not only contribute to the development of acute conditions but also lead to complications such as ear discharges, ruptured eardrums, and other issues.Additionally, they induce inflammation and pain in the middle ear [2].
Ear infections exert a considerable negative impact on health, serving as a significant contributor to avoidable hearing loss and representing a prevalent clinical issue [3].Globally, millions of children grapple with debilitating hearing loss, estimated by the World Health Organization (WHO) to affect over 5% of the population [3].A noteworthy aspect is that many instances of this condition are either curable or preventable.However, the escalating resistance to antibiotics displayed by these bacterial strains poses a growing challenge in successfully treating ear infections.This underscores the urgent need for research in this field to counteract resistance and safeguard public health.
Plant essential oils (EOs) are natural mixtures of abundant components such as terpenes, terpenoids, aromatic, and aliphatic constituents.The use of EOs as alternative for drugs was reported in many infection cases.Thyme oil were found to be the most effective agents against fungal isolates and they constitute a promising tool for the management of fungal infection causing the otitis media [4].According to a small study, drops crafted from a blend of essential oils extracted from cloves, English lavender, and herb-Robert demonstrated comparable effectiveness to an antibiotic ear drop, commonly prescribed for treating ear infections [5].
Nonetheless, some bacterial strains showed demonstrated heightened resistance to EOs following repeated treatment [6].Consequently, EO combinations exert a multifarious impact on microbial cells, rendering them potent antimicrobials.This characteristic not only expands the repertoire of potential natural antimicrobial agents but also offers an alternative to antibiotics, enhancing efficacy without fostering the development of resistance [7,8].
While some researchers have investigated the outcomes of blending Eucalyptus EOs with oils from different genera, comprehensive studies on combining different EOs from Eucalyptus are lacking.Notably, previous research has demonstrated synergistic activity against E. coli and S. aureus in binary mixes of E. globulus EO with those of Cinnamomum verum and Glycyrrhiza glabra [9].Additionally, diverse binary combinations of EOs from different Eucalyptus species yielded varying results against Paenibacillus amylolyticus and Bacillus cereus, including synergistic effects with Thymus vulgaris and Cinnamomum cassia Siebold, additive effects with Citrus reticulata (Blanco) M. Hiroe and E. globulus, and indifferent effects with Cinnamomum cassia Siebold and E. globulus [10].
In our earlier study, we we investigated the impact of eight different Eucalyptus EOs on bacterial strains associated with otitis [11].Notably, extracts from E. melliodora, E. bosistoana, and E. robusta exhibited potential antibacterial activity against both K. pneumoniae and E. coli.
Therefore, the primary objective of this study was to evaluate the antimicrobial efficacy of Eucalyptus EOs against specific bacterial strains implicated in otitis infections, considering various combinations.The central focus of the research lies in understanding the relationship between the chemical composition of these EOs and the antibacterial activity of both their binary combinations and combinations of binary combinations.

Plant material
Eight species of Eucalyptus L'Hér, which were collected in June 2017 from Tunisia, were used in our study.The clean and mature leaves were collected from the following species: E. accedens Fitzg, E. robusta Sm., E. punctata DC., E. melliodora, E. lesouefii Maiden, E. cladocalyx F. Muell, E. bosistoana F. Muell., and E. wandoo.Botanical voucher specimens were identified by Professor Mohamed Khouja Larbi and stored in the Pharmacognosy laboratory of the Faculty of Pharmacy in Monastir (Tunisia) under the following reference numbers: 0173, 0174, 0175, 0176, 0177, 0178, 0179, 180.

Extraction of essential oils
The EOs were extracted using an approved standard equipment specified by the European Pharmacopoeia.This involved hydrodistilling 100 g of coarsely crushed leaves for 4 h.Hydrodistillation was conducted in triplicate for each sample.The extracted EO was collected, dried with Na2SO4, and subsequently stored at 4 °C for analysis.

Gas chromatography (GC) and Gas chromatography-mass spectrometry (GC-MS) Analyses
The EO extracts were subsequently analyzed using GC and GC-MS in triplicate.The GC analysis was performed on a Hewlett-Packard 6890 apparatus equipped with an FID and an apolar HP5 capillary column.The experimental parameters were as follows: the oven temperature was programmed to start at 60 °C for 1 min, then gradually increased from 60 °C to 250 °C at a rate of 3 °C/min, and held isothermally at 250 °C for 3 min.The injector temperature was set at 250 °C whereas the detector temperature was set at 280 °C, and the used carrier gas was N2 at a flow rate of 1.2 mL/min.For each sample, 1 μL (diluted to 10% EO in purified hexane) was injected for analysis.The relative concentration was calculated using HP ChemStation software, which assimilates the percentages of peak areas to the percentages of various constituents.Retention indices (RI) were determined according to the retention time (Rt) of a series of n-alkanes (C9-C28).

Bacterial strains
The current study included three ATCC microorganisms [P.aeruginosa (ATTC 9027), S. aureus (ATCC 6538), and E. coli (ATCC 8739)] and three clinical bacterial isolates (H.influenzae, H. parainfluenzae, and K. pneumoniae).The ATCC strains were available in the culture collection of the Laboratory of Transmissible Diseases and Biologically Active Substances (Faculty of Pharmacy, Monastir, Tunisia), while the clinical strains were kindly provided by the Microbiology and Immunology Laboratory (EPS Farhat Hachad, Sousse, Tunisia).

Disc diffusion method
The Disc agar diffusion technique was used to assess the antibacterial activities of several EOs using bacterial inoculums of 0.5 McFarland; Mueller Hinton (MH) supplemented with 5% sheep blood.P. aeruginosa, E. coli, and S. aureus, however, did not undergo enrichment.The 90 mm inoculation plates were covered with absorbent discs (Whatman disc n°3, 6 mm diameter) that had been impregnated with 10 μL of each EO.. Every essay had a positive control disc containing 10 μg of Gentamicine ® .Upon incubation for 24 h at 37 °C, the inhibitory zones were measured and shown in millimeters.The findings were interpreted using the previously mentioned parameters [12,13]: diameter ≤ 8 mm indicated not sensitive or no inhibitory effect (-); diameter between 8 and 14 mm indicated sensitive ( +) or mild inhibitory effect; diameter between 14 and 20 mm indicated very sensitive or moderate inhibitory effect (+ +); diameter ≥ 20 mm indicated extremely sensitive or strong inhibitory effect (+ + +).Every experiment was conducted in triplicate, and the mean ± standard errors of mean was used to express the results.

Broth microdilution method
The Broth microdilution method was carried out to calculate the Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC) of the tested Eucalyptus EOs according to the National Committee for Clinical Laboratory Standard [14].
An overnight culture (37 °C) of each tested strain was prepared by adjusting the turbidity of each bacterial culture to reach an optical density of 0.5 McFarland standards.100 μL from each EO diluted in DMSO (10%), initially prepared at a concentration of 931 mg/mL, were added into the third well, followed by two-fold serial dilutions in MH Broth medium until the 12th well.Subsequently, 80 μL of MH, 10 μL of the inoculum, and 10 μL of 0.02% resazurin solution were added into each well.The skipped first and the second well were reserved for negative and positive controls, respectively.Negative control well contained bacteria only in the MH broth medium whereas, positive control well contained 10 μg/ mL of Gentamicin ® antibiotics.
After incubation for 24 h at 37 °C, the bacterial growth was characterized by color change from blue to pink.The MIC was defined as the lowest concentration that completely inhibits visible cell growth during 24 h incubation at 37 °C (blue colored well).To determine the minimum bactericidal concentration (MBC) values, 10 μL of each culture medium with no visible growth were removed and inoculated in MH plates.After incubation for 18-24 h at 37 °C, the number of surviving organisms was determined.The MBC was defined as the lowest concentration at which 99.9% of the bacteria culture were killed [15,16].As for other analyses, the experiments were performed in triplicate.The tested EOs are considered bactericidal if the ratio MBC / MIC < 4 and bacteriostatic if the ration MBC / MIC > 4.

Effect of the EOs blends on their antibacterial activities Combination's criteria
The two used methods (disc diffusion and broth microdilution) were carried out using two types of combinations: Binary combinations: The antibacterial effect of EO mixtures (1:1, v/v) was evaluated based upon their previously calculated inhibition zone diameter (izd) and according to the following choices: i) mixing previous none active EOs; ii) mixing EOs with no activity with those showing high activity; iii) mixing EOs with the most significant activity.

Combinations of binary combinations:
We used only the binary combinations that showed a izd value ≥ 15.00 mm; however, due to the small available quantity of the remaining EOs, we limit our study to 6 combinations of binary combinations.

Disc diffusion method
The antibacterial effect of EO mixtures (1:1, v/v) was evaluated based on their antibacterial activity expressed by their inhibition zone diameter (izd).
According to the izd values of EO mixtures, three effects could be observed [17]: Synergistic effect: when the effect of (A + B) > effect A + effect B; Additive effect: when the effect of (A + B) = effect A + effect B; Antagonist effect: when the effect of (A + B) < effect A + effect B; where A and B are two EO samples.
We performed 15 combinations (9 binary and 6 combinations of binary combinations) against 6 bacterial strains (total = 90 effects).Thus, the percentage of each type of antibacterial effect (synergic, additive, or antagonism) is calculated according to the following formula:

Broth microdilution method
The evaluation of the antibacterial effect is based on the fractional concentration indices (FICI), which allowed us to identify the type of interaction of the combined EOs against the tested bacterial strains.The FICI was calculated according to the following formula: [18] FICI = FICA + FICB, where FICA = (MIC of the combination (A-B) / MICA alone) and FICB = (MIC of the combination (A-B)/MICB alone).The results were interpreted as a total synergistic effect (FICI ≤ 0.5), a partial synergism effect (0.5 < FICI ≤ 0.75), an additive effect (0.75 < FICI ≤ 1), an indifference (1 < FICI ≤ 4), and an antagonism effect (FICI > 4).

Statistical analysis
The ANOVA test was used to compare the Eucalyptus species blends of EOs, the content of their chemical components, and the values of their izd against the tested bacterial strains.The level of statistical significance was determined at p < 0.05 using Duncan's multiple range tests.In order to identify the possibility of a relationship between the chemical composition of the EO mixtures and their antibacterial activities, all the values of the izd of bacteria growth inhibition were used for Principal Component Analysis (PCA) and Hierarchical Cluster Analysis (HCA) analyses using IBM SPSS Statistics for Windows, Version 23.0 (Armonk, NY: IBM Corp).

Disc diffusion method
The antibacterial activity of the Eucalyptus EO mixtures against six clinical and reference bacterial strains %Type_Effect = Number of effect type/Total number of effects * 100 exhibited significant variation among the EOs (p ≤ 0.001).Post hoc Duncan's multiple range tests identified six overlapping groups (a-f ) (Table 1).Assessment of the activities of EO mixtures, calculated in relation to the total number of observed effects, revealed that 12 combinations exhibited synergistic effects (13.33%), one combination (1.11%) had an additive effect, while the remaining combinations demonstrated antagonistic effects (85.55%).

Binary combinations
The results indicated that six out of nine binary combinations (bp, ac, bm, pm, pa, and pw) exhibited an increase in activity (Additive and Synergistic) against four bacterial strains (Haemophilus influenzae, Haemophilus parainfluenzae, Pseudomonas aeruginosa ATTC 9027, and Staphylococcus aureus ATCC 6538) (Table 1).
Specifically, the binary combination ac showed a synergistic effect against Pseudomonas aeruginosa ATCC 9027 and Staphylococcus aureus ATCC 6538, while the binary combination bm showed a synergistic effect against Haemophilus parainfluenzae and Pseudomonas aeruginosa ATCC 9027.The remaining binary combinations displayed an increase in the antibacterial activity against only one bacterial strain each.The bp combination showed a synergistic effect against Pseudomonas aeruginosa ATTC 9027; pw-synergistic effect against Staphylococcus aureus ATCC 6538.The sole additive effect was observed with the pa combination against Haemophilus influenzae (Table 1).

Combination of binary combinations
The results of the antibacterial activities of EO mixtures (Table 1) highlighted that four out of six combinations (bpac, bmpm, pwac, and pwpm) demonstrated a synergistic effect against two bacterial strains (Haemophilus influenzae and Haemophilus parainfluenzae) (Table 1).Notably, the pwac combination displayed a synergistic effect against both bacterial strains, while the combinations bmpm and pwpm exhibited a synergistic effect exclusively against Haemophilus influenzae.The remaining bpac combination demonstrated a synergistic effect solely against Haemophilus parainfluenzae.
While the majority of EO combinations displayed antagonistic effects against all or several bacterial strains, some exhibited notably higher antibacterial activity compared to individual or binary combined oils.For instance, the observed activity of binary combinations ac and bp against H. influenzae, pm, bl against H. parainfluenzae,

Principal Component's analysis (PCA) and Hierarchical Clusters Analysis (HCA) of the antibacterial activity
The PCA horizontal axis accounted for 47.30% of the total variance, while the vertical axis contributed an additional 20.96% (Fig. 1).Utilizing HCA based on the Euclidean distances, two distinct groups (A and B) of EO blends were identified by their antibacterial inhibition of growth with a dissimilarity ≥ 22.0% (Fig. 2).Subsequently, when the dissimilarity dropped to ≤ 10%, these groups were further subdivided into four subgroups (A1, A2, A3, A4, B1, B2, B3, and B4).Within the A4 subgroup, a subsequent division resulted in two subgroups (A4a and A4b) with a dissimilarity ≥ 7.0%.Conversely, the B4 subgroup underwent a division into two subgroups (B4a and B4b) with a dissimilarity < 6.0%.The horizontal axis effectively separated the EO combinations constituting the two main groups (A and B), while the vertical axis facilitated the separation of combined oils within the same groups.
Group A-It consisted of eight binary mixtures: pm, bm, pw, ar, pa, bp, ac, and wc.This group showed a positive correlation with axis 1 and a negative correlation with axis 2. These blends exhibited the strongest antibacterial effect against E. coli, S. aureus, and P. aeruginosa.
Subgroup A1-This subgroup comprises a blend of E. punctata and E. wandoo EOs (pw), displaying a potent inhibitory effect against S. aureus (23.30 ± 5.0 mm; izd).Despite exhibiting the highest activity against E. coli (14.7 ± 1.2 mm; izd) (Table 1) among all tested combinations, it was interpreted as antagonistic.Notably, this blend showed no activity against P. aeruginosa.Subgroup A2-This subgroup comprises a blend of E. punctata and E. melliodora (pm) as well as a blend of E. bosistoana and E. melliodora (bm).Both combinations share almost the same high activity against S. aureus and H. parainfluenzae, a mild effect against K. pneumoniae, and a low activity against E. coli.The separation between the two blends was essentially due to the difference in their activities against P. aeruginosa, which was interpreted as an almost moderate activity for bm blend and mild activity for pm blend.
Subgroup A3-This subgroup contains two binary types, ac and bp.It was distinguished by its moderate activity against P. aeruginosa, S. aureus, and H. influenzae and its mild activity against E. coli, H. parainfluenzae, and K. pneumoniae.The separation between the two binary EO blends was mainly due to the difference in their activities against S. aureus, which was interpreted as a strong inhibitory effect of the ac and mild activity for the bp blend oils.Subgroup A4a-This subgroup is constituted by the binary combinations ra and pa.These blends of oils showed low activity against K. pneumoniae and mild to moderate inhibitory effects against E. coli, P. aeruginosa, and H. parainfluenzae (8.3 ± 0.6 mm ≤ izd ≤ 14.0 ± 2.6 mm).However, they were differentiated by the values of their izd, which were significantly higher with ar blend oil than with pa blend oil, particularly against P. aeruginosa, H. parainfluenzae, and S. aureus; whereas a higher activity was shown for pa against H. influenzae.Subgroup A4b-This subgroup contains the binary combination wc.It formed a distinct dichotomy with subgroup A4a in the HCA and a separate subgroup in the PCA analyses, with a high negative correlation with axis 2. The wc combination showed the lowest activity against the majority of strains among all combinations.
This subgroup shared with subgroup A4a mild activity against H. influenzae and P. aeruginosa, H. influenzae, and S. aureus and the lowest activity against K. pneumoniae and E. coli.
Group B-This group is formed by the binary mixture of E. bosistana and E. lesouefii oils (bl) and also by the mixtures of six binary EOs such as bmpm, pwpm, paac, blbm, pwac, and bpac.They were characterized by the strongest activity against H. influenzae, H. parainfluenzae, and K. pneumoniae.Subgroup B1, reduced to a binary combination of E. bosistana and E. lesouefii essential oils (bl), which was clearly separated from the other subgroups of group B essential oil blends in both PCA and HCA analyses.It exhibited the highest activity against K. pneumoniae among the tested blends, and a moderate level of activity against H. parainfluenzae.Subgroup B2, limited to the bmpm EO blend.It exerted a high level of antibacterial activity against H. influenzae, almost comparable to that produced by Gentamicine ® .However, it was not active against P. aereuginosa.Subgroup B3, represented by EO blends of pwpm and acpa, were highlighted for their comparable activity to the antibiotic gentamicine against H. influenzae (29.0 ± 3.6 and 33.3 ± 2.9 mm; izd; respectively).However, these oils were found to be inactive against E. coli, P. aeruginosa, and S. aureus.They were also grouped together based on their mild antibacterial effect against K. pneumoniae.The main difference between these combinations is their activity against H. parainfluenzae (10.3 ± 0.6 and 15.3 ± 4.2 mm; izd; respectively).Subgroup B4 is formed by three EO blends: bpac, pwac, and bmbl.These blends are characterized by strong activity against H. influenzae and H. parainfluenzae, with izd varying from 20.0 ± 0.0 to 30.7 ± 1.2 mm.However, their activity is still lower than that produced by the antibiotic gentamicine.A synergistic effect is observed with the pwac blend against both strains, and with the bpac against H. parainfluenzae only.Despite the antagonistic effect produced by the association of bpac essential oils with H. influenzae, the activity is still classified as strong (28.7 ± 5.5 mm, izd), comparable to that produced by gentamicin (31.4 ± 2.1 mm, izd).A moderate level of activity was shown for pwac, bpac, and bmbl combinations (15.0 ± 4.0, 12.0 ± 0.0 and 12.7 ± 1.2 mm; izd; respectively) against K. pneumoniae.

Broth microdilution method
The results of the MIC and MBC of the tested EO blends are presented in Table 2.They demonstrated bactericidal effects against the bacterial strains tested, with MBC/ MIC values less than 4, except for the pm blend against K. pneumoniae, the mbpm blend against S. aureus, and the paac blend against P. aeruginosa, E. coli, and H. influenzae.The binary EO combinations pa and pw exhibited the strongest antibacterial activity against all tested bacterial strains.
The pm and pw combinations showed the lowest MIC and MBC against K. pneumoniae and H. parainfluenzae (0.8 mg/ml, 3.3 mg/ml and 0.8 mg/ml, 1.6 mg/ml, respectively).The pa blend also showed low MIC and MBC against K. pneumoniae (6.5 mg/ml) and E. coli (14.1 mg/ml), whereas the pm blend exhibited similar results as the pa blend.
The highest MIC value (415.5 mg/ml) was observed for the binary mixture of bp, ac, and blbm against P. aeruginosa.However, the highest MIC results were observed with the blend of ar against H. influenzae and H. parainfluenzae, the paac blend against S. aureus, and the ac blend against E. coli.

Binary combinations
The results of the antibacterial activities of EO mixtures indicated that seven (pa, pw, pm, bl, bp, ar, and wc) out of nine binary combinations showed an increase in activity (total synergism, partial synergism, additive effect) against three bacterial strains (E.coli, H. parainfluenzae, and K. pneumoniae) (Table 1).
The pw blend oils demonstrated the best antibacterial activity, displaying total synergism against H. parainfluenzae, E. coli, and K. pneumoniae (FIC I of traces, 0.3, and 0.4, respectively).Additionally, the pa blend showed the highest activity against E. coli and K. pneumoniae (FIC I = 0.2 for each).The later bacterial strain was sensitive to pm (FIC I = 0.2).
The lb blend showed a partial synergistic effect against K. pneumoniae (FIC I = 0.7), while the bp, ar, and wc blends showed an additive effect against H. parainfluenzae, P. aeruginosa, and E. coli, respectively (Table 3).

Combination of binary combinations
The results of the study showed that none of the binary combinations had a synergistic effect.However, a significant additive effect was observed for bpac and bmpm against Gram negative bacteria such as P. aeruginosa; for paac against H. parainfluenzae; and for bmpm against Gram-positive bacteria such as S. aureus (Table 3).

Chemical Principal Component's analysis (PCA) and Hierarchical Clusters Analysis (HCA)
In order to investigate the relationship between the chemical composition and antibacterial activity of EO blends, 21 selected oil blend components listed in Table 4 were analyzed using PCA and HCA.The PCA analysis showed that the horizontal axis explained 31.32% of the total variance, while the vertical axis explained 22.31% (Fig. 3).The HCA analysis revealed two distinct groups of essential oil blends (I and II) with a dissimilarity score greater than 17 (Fig. 4).Axis 1 was responsible for separating the essential oil blends of group I from those of group II, while axis 2 was responsible for separating EO blends within group II (Fig. 4).
Group II showed a high correlation with axis 1 and a low correlation with axis 2. With a dissimilarity index greater than 9, it was separated into four subgroups: IIa, IIb, IIc, and IId.
Subgroup IIa, limited to the mixture of E. wandoo and E. cladocalyx oils (Wc).It shared with group IIb (ac, pwac, paca, and bpac) the highest mean percentage in globulol, methyl amyl acetate, aromadedrene, viridiflol [7], and epiglobulol, as well as medium percentages in 1,8-cineole, α-pinene, and p-cymene, and the lowest content in β-pinene.The separation between the EO blends of the two groups was mainly due to the variation in their chemical component content.In fact, the wc blend oils were very close to the ac blend oils in both the HCA and the PCA, which could be explained by their similar high mean percentages of globulol and methyl amyl acetate.However, they were separated by the increasing level of p-cymene in the wc blend oils and its decreasing level in the ac blend oils, which were significantly richer in α-pinene.This compound was identified as    Subgroup IIc, formed by the EO blends of bl, bm, and blbm.These oils are distinguished by their highest levels of 1,8-cineole, spathulenol, terpinene-4-ol, and α-terpineol, as well as their relatively high contents of α-pinene and trans-pinocarveol.However, they are relatively poor in p-cymene, rosifoliol, methyl amyl acetate, and cuminaldehyde.The PCA and HCA analyses showed that bm and blbm blends were similar in their disposition, with the highest content of 1,8-cineole and low content of β-pinene, compared to lb blend oil, which is richer in spathulenol.
Subgroup IId, constituted by pa, bp, pm, bmpm, pwpm, and pw blends.Their EOs were characterized by the highest mean percentage in p-cymene, trans-pinocarveol, cryptone, p-cymen-8-ol, and a relatively high mean percentage in β-pinene.With dissimilarity greater than 7, pa blend oils formed a deep dichotomy within the rest of the subgroup IIb blends oils in the HCA analysis.This separation was essentially explained by its disposition, the lowest mean percentage in 1,8-cineole and relatively the highest content in α-pinene.On the other hand, the differentiation of bp, pm, bmpm, pwpm, and pw blends was primarily affected by their content in 1,8-cineole and p-cymene.The pw and pwpm blends of oils had the highest content in p-cymene, which distinguished them from the other blends, bp, bmpm, and pm, which had relatively higher content in 1,8-cineole.This separation was evident in both principal component analysis (PCA) and HCA, where pw and pwpm blends oils correlated negatively with axis 1 and were clearly separated from the other blends.

Discussion
The antibacterial activities of EOs are often attributed to the complexity of their chemical composition as well as to the synergistic, additive, or antagonistic effects of their chemical components [19].Therefore, we tried to enhance the antibacterial activity by mixing EOs.In general, the antibacterial activity of the combined EOs varied significantly according to the chemical composition, the bacterial strains, and the method used.To find a logical explanation for this variation, we based our analysis on the principle of the quantitative and qualitative addition of the main compounds constituting the EO blends (Table 4) and their correlations with the observed activity.

Correlation of the antibacterial activity of EO blends tested by the disc diffusion method with their chemical compositions
It was reported that the EO's biological and pharmacological activity is attributed to the effect of the complex Fig. 3 PCA of twenty one components for the 15 Eucalyptus EOS blends extracted from leaf of eight Tunisian Eucalyptus species.For the abbreviation of the Eucalyptus species (▲), see Table 1 interaction between the EO components' functional groups belonging to some chemical classes such as phenols, alcohols, esters, ethers, and to their mean percentages and the bacterial strains [8,20,21].
Therefore, the combination of these two results allowed us to deduce that the increased activity against H. influenzae could be attributed to a synergistic effect between globulol, viridiflorol, methyl amyl acetate, α-pinene, and p-cymene as well as the average percentage of 1,8-cineole.However, the antagonistic effect could be attributed to the dominance of 1,8-cineole, which, by interacting with other compounds such as trans-pinocarveol, α-terpineol, and β-pinene may enhance the antibacterial resistance.These observations were supported by several studies, which reported that Eucalyptus oils rich in 1,8-cineole and other major components exhibited weak antimicrobial activity; however, the presence of a medium content of 1,8-cineole, with other terpenes such as globulol, α-pinene, and p-cymene in Eucalyptus oils could produce a synergistic or additive effect [22,23].It was also reported that the hyrodrphobic terpenes, α-pinene, p-cymene had a low water solubility and low hydrogen-bonding capacity, which resulted in a lack of their antibacterial activity [24,25].However, these compounds could be accumulated in cell membranes, disturbing their integrity as well as increasing their permeability [18].Therefore, it could facilitate the transfer of the active components, such as the terpenoids, and enhance the antimicrobial activity.
Therefore, the inactive compounds might influence resorption or the kinetics of the reactions, modulating the biological activities of the tested compounds.It is worth noting that the combination of both major and minor components can modify the biological activity of the EOs and exert a significant synergistic or antagonistic effect [27,28].
Comparative analysis of the discussed above results suggests that the synergistic effect observed against P. aeruginosa may be mainly due to the interaction between 1,8-cineole, aromadendrene, and globulol; whereas the presence of a relatively high mean percentage of p-cymene, γ-terpinene, cryptone, and methyl amyl acetate could significantly reduce antibacterial activity.
It was reported that the E. gobulus oil fruit rich in 1,8-cineole, globulol, and aromadendrene did not show a substantial inhibition against the Gram negative bacterial strains P. aeruginosa, K. pneumoniae, and E. coli.However, combinations of aromadendrene and 1,8-cineole could show an additive or synergistic effects against Gram positive bacteria, when the dose of 1,8-cineole is significantly higher than that of aromadendrene [29].

Escherichia coli
The analysis of the antibacterial activity revealed an antagonistic effect of all tested oil blends against this bacterial strain.For instance, the combination of E. robusta oil (r), known by its relatively high content in citronellal, α-terpineol, endo-borneol, and rosifoliol and a high activity against E. coli, with E. accedens oil (a), known by its lower content of above components and a significant higher mean percentage of α-pinene and almost an equal amount of 1,8-cineole, showed a significant reduction of the antibacterial activity of their mixture (ra blend); showing antagonistic effect.
The most probable explanation for this antagonism is that a high concentration of the monoterpene hydrocarbon α-pinene, reduces aqueous terpene solubility, affecting the antimicrobial activity of the active components such as alcohols α-terpineol, endo-borneol, and rosifoliol [11,24,30,31].It was reported previously that the aldehyde component, citronellal, is inactive against E. coli, which had also a low water solubility [30], which could affect the solubility of the active components and support this antagonistic effect exerced by the high content of α-pinene.
All the above discussed results allow us to support the point of view that the antagonistic effect exerced by the hydrophobic mononterpens hydrocarbons α-pinene and p-cymene could reduce the solubility of the oxygenated compounds and therefore affect their biological activities [32].

Klebsiella pneumoniae
As discussed for E. coli, the analysis of the antibacterial activity revealed an antagonistic effect of all tested oil blends against this bacterial strain.The wc blend exhibited an antagonistic effect against K. pneumoniae.The comparison of the wc EO chemical composition to that of E. cladocalyx (c), which showed a strong inhibitory effect against this strain, revealed that the wc blend has almost the same content of 1,8-cineole, a higher content of p-cymene, and a lower mean percentage of globulol, epiglobulol, rosifoliol, viridiflorol, aromadendrene, and methyl amyl acetate.
These results suggest that the decreased antibacterial activity could be explained by the reduction in the mean percentage of active compounds, such as the terpenoids, and by a high concentration of the monoterpene hydrocarbons p-cymene.Despite the antagonistic effect shown for the lb blend against the same strain, this activity was significantly increased compared to those produced by the single oils.This difference could be influenced by the difference in the concentration of several compounds involved directly or indirectly in the antibacterial activity.For example, when we compare the chemical composition of the blend lb to that of the single oil of E. bositoana (b), which showed a strong activity, we found a small decrease in the concentration of 1,8-cineole, α-pinene, and aromadendrene, an increasing level of terpinen-4-ol, trans-pinocarveol, α-terpineol, globulol, β-pinene, p-cymene, and almost the same concentration in spathulenol.We suggest that the increased antibacterial activity could be due to the increasing level of the oxygenated terpenes such as the monoterpene alcohols terpinen-4-ol, trans-pinocarveol, and α-terpineol which were linked to their high antibacterial activities against several Gram negative bacteria such as K. pneumoniae [33,34].Additionally, it was reported that the combination of the two isomers terpinen-4-ol and α-terpineol exhibited a synergistic effect on the Gram negative bacteria Shigella flexneri [35].This activity could be supported by the presence of the sesquiterpenic alcohols spathulenol, globulol, as well as by the main compound 1,8-cineole.The later was reported as the principal components responsible in the antibacterial activity against K. pneumoniae [36,37].
However, the monoterpenes hydrocarbons β-pinene, p-cymene, and α-pinene could interact together by reducing the water solubility of the terpenoids, such as the 1,8-cineole, which known by its low water solubility, and considerably reduces the effect of the active compounds [24].Therefore, the blend lb showed a slightly increase of its antibacterial activity, compared to that observed with E. bositoana oil.
These findings suggest that compounds such as epiglobulol, viridiflorol, globulol, methyl amyl acetate, aromadendrene, and other minor components may be responsible for the antagonism effect.In fact, these compounds were reported as active against a wide range of bacterial species, including S. aureus [24,29,38].However, their combination could have an antagonistic [8].
This mechanism of interaction is not well understood and requires further investigations.It could be explained by an interaction between these compounds to reduce the active terpenes solubility, or by competing for the same binding sites on bacterial active sites [8,19].

Correlation of the antibacterial activity of EO blends tested by the broth microdilution method with their chemical compositions
The results showed a synergistic effect of certain binary EO blends, essentially against Gram negative bacterial strains such as E. coli, K. pneumoniae, H. influenzae, and H parainfluenzae.However, none of the combined of binary blends has shown synergistic.These results were in concordance with those reported by Kachkoul et al. [39].
The active binary blends (pw, pm, pa, bp, and bl), were composed of single EOs with low antibacterial activity, such as E. punctata (p) and a medium or high activity, such as E. wandoo (w), E. melliodora (m), and E. accedens (a).These blends, except bl blend, are belonging to the same chemical group according to the ACP analysis (group IIc) and sharing together the highest mean percentage in monterpenic hydrocarbons, monoterpenic alchols, monoterpenic aldehydes, and ketones.All of these chemicals are represented essentially by p-cymene, trans-pinocarveol, cuminal, and cryptone, respectively.They are characterized by a medium content in 1,8-cineole.However, bl, bm, and blbm blends, belonging to another chemical group, characterized by their high content in 1,8-cineole, terpine-4-ol and lower content in p-cymene and cryptone, cuminal and p-cymen-8-ol, showed an antagonistic effect against E. coli, K. pneumoniae, H. influenzae, P. aeruginosa, and H. parainfluenzae.
The results suggest that the presence of a relatively low to medium content in 1,8-cineole and a medium to high mean percentage in p-cymene, as well as the presence of cryptone, cuminal, and p-cymen-8-ol produced a synergistic effect against these strains.It was reported that terpenic components can interact to either decrease or increase antimicrobial activity, depending upon their relative concentrations and the overall susceptibility of the target microorganism.Moreover hydrophobic terpenes such as p-cymene and α-pinene are expected to be accumulated preferentially in the cell membranes to facilitate the absorption of the active compounds such as 1,8-cineole, shown to be active against E. coli [26,40].Another study reported that hydrophilic EO components such as cryptone and p-cymen-8-ol showed a high hydrogen binding, promoting the entrance of hydrophilic components; whereas hydrocarbon terpenes, such as limonene, provide higher enhancing activity for the lipophilic components [41].

Comparison between disc diffusion, broth microdilution methods and their activities against the bacterial strains
We highlight in the present study a discordance in the antibacterial activity results obtained by the disc diffusion and the broth microdilution methods.This observed discordance could be related to several factors: i) the low diffusion ability of some active chemical compounds through the agar, which in itself is highly dependent on their polarity and on water solubility; ii) the chemical structure of the EOs' components and their molecular properties; iii) their site and mode of action, and their interaction with the surrounding environment [19,24].Some differences in the obtained results of the present study could be attributed to the tested bacterial strains and the chemical compositions of EO blends.
In our study, three binary EO blends (pm; pw, and ac) showed higher antibacterial activity against Gram-positive bacteria (S. aureus) using disc diffusion method than against Gram-negative bacteria.However, the later was more sensitive to five EO blends (bl, bp, pa, pm, and pw) using broth microdilution method (Table 5.supl).
For example the blends pw and pm, which differed significantly in their content in 1,8-cineole and sharing almost the same mean percentage in monoterpene alcohols trans-pinocarveol, α-terpineneol, terpinen-4-ol, p-ymen-8-ol, had a synergistic effect against S. aureus by the disc diffusion method, however they showed an antagonistic effect by the broth microdilution method.This difference in antibacterial effects was also observed with ac blend, characterized by its high mean percentage of the sesquiterpene alcohols globulol, epiglobulol, viridiflorol, and the ester methyl amyl acetate.
We suggest that the activity could be due to the presence of monoterpene and sesquierpenes alcohols and their interactions, as they are known by their high water solubility and high affinity to cell membrane of bacterial strains [42].
Additionally, the Gram(-) bacteria is constituted by a lipopolysaccharide layer (LPS) which acts as a barrier for macromolecules and hydrophobic compounds, such as those present in the above discussed chemical components [43,44].Therefore, the Gram (-) bacteria are more resistance against the hydrophobic active components than the Gram-positive bacteria.Indeed, the hydrophobicity properties of EO components and the synergistic action of their components enable them to accumulate in cell membranes, which can change the fluidity, disturb the structures and cause the increase of the cell membrane penetrability [45].
In another hand, the Gram( +) bacteria sensitivity to some EO components could be attributed to the membrane structure, as they lack the LPS layer [46].

Conclusion
To the best of our knowledge, this study marks a pioneering demonstration of the antibacterial properties of Eucalyptus sp.EOs combinations against the microorganisms responsible for ear infections.Our investigation uncovered an enhancement in the efficacy of these EOs when combined at specific proportions, albeit not all potential combinations were explored.
The effectiveness of these essential oil blends exhibited variation depending on the targeted microorganism and the diverse chemical constituents within the mixture.These versatile combinations present an alternative to conventional medications, which are progressively becoming less effective against numerous microorganisms responsible for significant ear infections.The broad array of chemical ingredients and potential synergistic effects among compounds make these combinations promising in the treatment of such infections.

Table 1
(continued)bl against K. pneumoniae, and bp against S. aureus surpassed the activity of the individual EOs used.Interestingly, certain combinations of binary blends demonstrated even higher activities than those observed with binary EO blends.Notable examples include bpac against H. influenzae and blbm against H. influenzae and H. parainfluenzae.

Table 2
Minimal inhibitory concentration (MICs), minimal bactericidal concentrations (MBC s), and MBC/MIC ratio for 15 blends Eos extracted from leaves of eight Eucalyptus Species

Table 4
Content [%] of the 21 Compounds selected for the Principal Component and the Hierarchical Cluster Analyses in 15 blends of EOs Extracted from the Leafs of eight Eucalyptus species and their chemical classes